Project Summary/Abstract Despite a decade of work on super-resolution imaging, a large amount of sub-cellular phenomena remains in- visible due to the poor temporal resolution of current techniques. As a result, there is an urgent need for a ?exible, accessible, and quantitative super-resolution imaging system with improved temporal and spatial reso- lution. For example, DNA has characteristic lengths between 5 nm and 150 nm that exhibit signi?cant dynamic changes over time scales as small as 1 ms, which are length and time scales that are not within the capabilities of current super-resolution methodologies. Our long-term goal is to develop and apply measurement tools to further our understanding of mechanisms and dysfunction of sub-cellular phenomena. The overall objective of this application and the next step in attaining this long-term goal is to drastically improve the temporal resolu- tion of super-resolution through both single-acquisition super-resolution microscopy as well as 3D localization at an order of magnitude greater ?uorophore density. The rationale underlying this research is that current super- resolution instruments are inadequate for visualizing many small-scale and fast sub-cellular processes. Reaching these smaller lengths and faster time scales requires a new approach toward visualizing these phenomena if we are to understand them with suf?cient ?delity to treat or diagnose diseases. To ensure that we achieve the ob- jective of this application we have developed the following Speci?c Aims: 1) Development of Single-Acquisition Super-Resolution Microscopy, which will result in a 40% improvement in diffraction limited resolution in every im- age, and 2) Development of 3D Dense-Emitter Image Analysis Algorithm, which will result in the ability to either locate in 3D emitters as close together as 60 nm with 10 nm isotropic resolution from a single image or recon- struct the 3D sub-diffraction density of closer emitters. Together these new and improved capabilities will enable improvements to the temporal resolution with which super-resolution data is acquired. The approach is innovative in our opinion, because it utilizes a novel imaging system (Bessel Beam Microscopy) that trades low-frequency information for spatial resolution, which differs from current super-resolution systems that trade temporal reso- lution for spatial resolution. The proposed research is signi?cant because it expands super-resolution imaging capability in an area where there is a particularly urgent need. Providing high-speed sub-diffraction limit 3D re- construction capabilities will allow real-time monitoring of, for example, chromatin packing level and stiffness in response to transcription. Additionally, stochastic super-resolution techniques such as STORM/PALM will bene?t from the accurate localization at dense labeling, improving their temporal resolution.